Xylose uptake was stimulated rapidly when rat soleus muscles were incubated in Krebs-bicarbonate medium made hyperosmolar by the addition of 200 mM mannitol. The stimulatory effect of hyperosmolarity increased as the concentration of mannitol was raised from 50 to 200 mM; above 200 mM, the effect tended to decline. Mannitol stimulated sugar transport submaximally compared with the effect of insulin. The stimulatory effect of hyperosmolarity persisted over a 60-min incubation period, but could be reversed by transferring the muscles to an isotonic incubation medium. Xylose uptake measured in the presence of insulin (0.1 U/ml) was depressed when muscles were exposed to concentrations of mannitol greater than 100 mM. This effect was not reversible; xylose uptake remained depressed when these muscles were transferred to an isotonic medium. Hyperosmolarity also depressed the binding of 125I-insulin irreversibly. These inhibitory effects of hyperosmolarity were associated with the lowering of muscle ATP. This effect on ATP provides an explanation for the inhibitory effects of hyperosmolarity on insulin-stimulated sugar transport and on insulin binding, in terms of the ATP-dependence of these two processes.